resveratrol: challenges in translation to the clinic · 29/10/2010 · now that 60 minutes,...

1 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Copyright © 2010 American Association for Cancer Research

Resveratrol: challenges in translation to the clinic

A critical discussion

Lalita Subramanian, Sherry Youssef, Saswati Bhattacharya, Jason Kenealey, Arthur S.

Polans and Paul R. van Ginkel

The University of Wisconsin Carbone Cancer Center, the Department of Ophthalmology

and Visual Sciences, the UW Eye Research Institute, and the Department of

Biomolecular Chemistry, University of Wisconsin, Madison 53792

Corresponding Author: Paul van Ginkel, Department of Ophthalmology and Visual

Sciences, University of Wisconsin School of Medicine and Public Health, Room K6/454

Clinical Sciences Center, 600 Highland Avenue, Madison, WI 53792. Phone: 608-265-

5409; Fax: 608-265-6021; E-mail: [email protected]

Statement of Translational Relevance

Side effects from chemotherapy highlight the need for effective non-toxic anti-cancer

agents. Resveratrol has consistently proven effective at tumor inhibition in diverse

human cancer models, while remaining non-toxic. This suggests that resveratrol could

pioneer a novel class of chemotherapeutics. It already is being extensively promoted in

the unregulated nutraceutical sector despite recent controversies regarding target

interaction, bioavailability and other essential matters. However, fundamental aspects of

Published OnlineFirst on November 2, 2010 as 10.1158/1078-0432.CCR-10-1486

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resveratrol action need to be understood before it can be developed into a clinically

viable anti-cancer drug. We present a short but critical discussion of these issues and

suggest important experiments that could pave the way to the successful translation of

resveratrol to the clinic.

Abstract

Low cancer survival rates and the serious side effects often associated with current

chemotherapeutics highlight the need for new and effective non-toxic anti-cancer

agents. Since 1997 when Jang et al. first described resveratrol’s ability to inhibit

carcinogenesis, it has consistently proven effective at tumor inhibition in diverse human

cancer models. This has raised the hope that resveratrol would pioneer a novel class of

non-toxic chemotherapeutics. As a consequence of initial basic and pre-clinical studies,

resveratrol is now being extensively promoted in the unregulated nutraceutical sector.

However, some fundamental aspects of resveratrol’s action need to be understood

before it can be developed into a clinically viable anti-cancer drug. These pertain to the

key mechanism(s) by which resveratrol potentiates its anti-tumor effects. Current

research suggests that these might be through novel pathways requiring an

understanding of cellular uptake, sentinel targets and in vivo biological networks. The

metabolism of resveratrol and its bioavailablity also warrant further consideration in light

of recent in vitro and in vivo studies. Finally, we need to appreciate the sorts of

information about resveratrol that may translate between different disease entities. We

present a critical discussion of these issues and suggest important experiments that

could pave the way to the successful translation of resveratrol to the clinic.






Now that 60 minutes, Barbara Walters, National Public Radio, Dr. Oz and a

myriad of columnists at the New York Times and other papers have offered their

comments and recommendations regarding the health benefits of resveratrol, including

its potential anti-cancer properties, perhaps it’s time for scientists to ask a few essential

questions about the popular product before considering a further pitch to the public.

In 1997 Jang et al. published their seminal paper on resveratrol’s ability to inhibit

the three major stages of carcinogenesis (1). Since then, there has been an explosion

of literature about resveratrol’s effects on cancer. Some fundamental aspects of our

understanding of resveratrol, however, are yet to be resolved. These gaps in our

knowledge must be addressed before the full potential of this non-toxic compound as an

anti-cancer drug can be realized.

I. Cellular uptake

What evidence do we have that resveratrol actually enters cancer cells?

A critical first step to delineate the mechanisms of drug action is to determine

whether resveratrol mediates its effects by cell surface receptors or intracellular targets

in tumor and other cells. Both intra and extracellular resveratrol targets have been

proposed (see section II). However, a direct binding partner has yet to be convincingly

established. Elucidation of whether resveratrol enters cells or acts at the cell surface will

shed some light on the targets on which it acts.

Resveratrol is a hydrophobic compound and currently the only compelling

evidence for entry of resveratrol into cells comes from highly specialized cell types:






intestinal and liver cells. When a monolayer of colonic Caco-2 cells was treated in vitro

with resveratrol, a saturating dose-dependent apical to basolateral transport was

observed (2, 3). In another study, the human hepatoblastoma cell line HepG2 and

human hepatocytes, were used to compare resveratrol transport in normal and tumor

cells (4). Resveratrol uptake was detected by its intrinsic fluorescence properties as well

as by using radiolabeled drug. Concentration- and temperature-dependence of uptake

were examined and results indicated that both passive diffusion and carrier-mediated

transport might be involved.

The above cell types, however, have specialized uptake mechanisms. The

intestinal epithelium is adapted for absorption of nutrients from food. A study of drug

transport using a Caco-2 monolayer may indicate transepithelial transport of a drug, but

does not necessarily imply that other cells have similar uptake mechanisms. Similarly,

one of the main functions of hepatocytes is to extract protein-bound drugs and

metabolites from circulation. Consequently, hepatocytes are equipped with many

different transport proteins in the basolateral membrane. The only other evidence of

possible resveratrol uptake has been shown indirectly through sulfotransferase 1A1

activity associated with two breast cancer cell lines resulting in the conversion of

exogenously added resveratrol to resveratrol 3-O-sulfate (5). These studies have not

been replicated in other tumor cells.

If resveratrol cannot be shown to efficiently enter all tumor cells, it may be more

likely that its anti-tumor effect is the result of binding to an extracellular target.

Therefore, further studies are necessary to directly address cellular uptake in cancer

cells. New technology, such as two-photon microscopy, may be particularly appropriate






for detecting the real-time uptake of unmodified resveratrol and its metabolites by taking

advantage of their intrinsic fluorescence under conditions of minimal photo-bleaching.

Results of such studies will indicate the validity of intracellular targets identified thus far

and aid in the identification of new sentinel targets.

II. Targets — fast and slow

What are the direct targets of resveratrol and can we differentiate binding events

from indirect effects of resveratrol?

The wealth of resveratrol literature is in part a result of the myriad of pathways

and molecules that are altered in response to resveratrol treatment (reviewed in 6).

Some of these effects may be cell type specific, tissue type specific or depend on

whether a cell is transformed. Through parametric analysis of gene set enrichment

(PAGE analysis), resveratrol has been found to cause a significant alteration in 127

pathways (7). Certain effects of drug treatment are measurable in a matter of seconds

(8), whereas others only become apparent after hours or even days. This then brings up

the question which of these effects is a direct result of resveratrol binding and which are

downstream effects of the initial target interaction.

Several molecular targets have been postulated as mediators of resveratrol’s

anti-cancer effects (7). Approaches such as a phage-display screen using biotinylated

resveratrol identified several candidates (9). However, the direct binding of resveratrol

to these targets in a cellular context has yet to be convincingly demonstrated.






An example of a direct binding target identified through column chromatography

is quinone reductase 2 (10). The binding constant for NQO2 was in a physiologically

relevant range. However, not all tumor cells have measurable levels of this protein

despite having similar sensitivity to the drug (11). Therefore, binding to this protein may

not be part of a general mechanism for resveratrol anti-tumor action.

Other targets reported include DNA polymerase α, PKC, PKD, COX2,

ribonucleotide reductase, and F0/F1 ATPase/ATP synthase based on alterations of

enzymatic activity. Only F0/F1 ATPase/ATP synthase however has been shown to

interact directly with resveratrol. An additional complication in identifying and validating

such targets is that binding and enzymatic experiments in vitro or in cells in culture may

not reflect in vivo conditions. Since levels of unmetabolized resveratrol in vivo don’t

exceed the low micromolar range (11), it is possible that some observed target binding

and modulations of pathways are only induced because of exposure of isolated

enzymes or cells to constant and high drug concentrations in vitro.

A more recent group of potential targets, the sirtuins, has been the subject of

intense research as mediators of resveratrol’s life extending activity as well as anti-

tumor activity. The direct binding of resveratrol to sirtuins has not been shown. Early

work showing direct resveratrol activation of purified SIRT1 using a fluorescently

modified substrate for sirtuin was shown to be an artifact of the assay (12). Additionally,

the effects of resveratrol on sirtuins in cells appear to be measureable only after several

hours. This is suggestive of downstream or indirect effects, if at all. More recent studies

also conflict with the original observation that resveratrol has an effect on Sir2 activity in

vivo (13) or an effect on the lifespan of yeast (13), Drosophila, or C. elegans (14).






Extracellular receptors for resveratrol such as insulin growth factor receptor

(IGFR) and integrin αVß3 have been proposed. The IGFR study compared downstream

activity using known ligands (15). No direct binding was shown in this study. The αVß3

study relied on competition assays using a known ligand and binding studies using

membrane fractions (16). Radiolabelled resveratrol was used to suggest direct binding

to the ß3 subunit of the integrin. However, given its properties, resveratrol is likely to

associate with membranes readily, making it difficult to assess the specificity of these

binding studies. Even though further work is needed to establish that resveratrol binds

to these extracellular receptors, these studies suggest that resveratrol could activate

several extracellular targets.

One of the very early in vitro events observed in tumor cells following resveratrol

addition is an increase in intracellular calcium and can be measured within seconds

after drug treatment (8). Calcium signaling (Fig. 1) may be a shared early response

underlying the non-toxic feature of different anti-cancer natural products (17). If so, it

may be possible to define a novel calcium-based mechanistic model for the

development of a new generation of non-toxic chemotherapeutics using resveratrol as a

model compound. However, the sentinel targets involved in calcium activation are yet to

be identified. Further, we have to recognize that some binding targets for resveratrol

activate early events while others might potentiate later events.

In spite of important progress, the binding targets of resveratrol that mediate its

anti-cancer activity remain unknown. The relevant targets mediating effects in vitro

versus in vivo might differ, and there appear to be several targets within a single cell

type as well as differences between cell types. To better understand the mechanisms






through which resveratrol mediates its anti-cancer properties, studies need to better

reflect conditions in vivo at drug levels that lead to inhibition of tumor growth.

III. Resveratrol Actions

Is resveratrol anti-proliferative, pro-apoptotic and anti-angiogenic?

In vitro studies of resveratrol using a variety of tumor cell lines have shown the

drug to be anti-proliferative as well as pro-apoptotic, through the activation of many

different pathways. Resveratrol inhibits cell cycle progression by modulating major cell

cycle regulators, arresting cells at the G1/S, S or G2/M phase and by inhibiting DNA

synthesis. These effects, however, have not been demonstrated in vivo. The

mechanisms causing tumor inhibition therefore are unclear even though tumor inhibition

is observed in many different animal models.

Alternatively, it is well established that angiogenesis is critical to tumor

progression. Resveratrol has been reported to have an anti-angiogenic effect in certain

tumor models as indicated by decreased vessel density in treated tumors (18-21).

However, an observed decrease in microvessel density cannot differentiate between

cause and effect. Either the inhibition of tumor growth results in fewer blood vessels or

an anti-angiogenic effect causes decreased tumor outgrowth due to a lack of nutrients

and oxygen. The tumor-inhibitory effects in vivo could be due either to direct effects of

resveratrol on tumor cell growth or indirect effects on angiogenesis. An initial study in a

mouse model of Lewis Lung Carcinoma indicated a resveratrol effect on

neovascularization (19). However, more in vivo studies are needed to show a direct






effect of resveratrol on blood vessel formation as the mechanism for tumor inhibition

using cell-specific reporter systems.

In contrast to tumor inhibition, higher local doses of resveratrol result in massive

tumor cell death and tumor regression as indicated by TUNEL staining and histology

(11, 22). Thus the reported pro-apoptotic effects of resveratrol may only be relevant

when local concentrations of resveratrol more closely match the higher concentrations

studied in vitro. Therefore, strategies to improve the bioavailability of resveratrol either

systemically or locally will provide more potent anti-cancer effects.

IV. Bioavailability

How do we reconcile the anti-cancer effects of resveratrol determined from in

vitro studies with in vivo measurements of resveratrol bioavailability?

Preclinical studies using resveratrol in animal models of different human cancers

indicate that resveratrol has potent anti-tumor properties. However, measurements of

serum levels of unmetabolized resveratrol suggest that its bioavailability is very low after

oral or intraperitoneal administration of the drug. This is mainly due to poor absorption

and ready metabolism to glucuronidated and sulfated compounds, followed by rapid

clearance of these metabolites. Serum levels of unmetabolized resveratrol peak in the

sub to low micromolar range (<3 µM) within minutes of oral drug administration, and

decrease rapidly thereafter (11, 23-26). One implication of these findings is that the

direct activation of cellular targets by resveratrol needs to reflect this transient

occurrence of both resveratrol and the metabolites. In addition, no accumulation of






resveratrol in tumors or normal tissues has been found after extended treatment

regimens (11). These data contrast sharply with in vitro studies wherein sustained

concentrations of tens of micromolars are required to obtain substantive anti-tumor

effects. This apparent conundrum was discussed elegantly by Gescher and Steward

(27). These observations may suggest one of two different things: 1) the mechanisms of

action of resveratrol are different in vivo and in vitro; or 2) the in vitro conditions for

growing cells somehow alter the sensitivity to resveratrol. In the first case, the anti-

angiogenic activity of resveratrol could indirectly inhibit tumor growth. Such properties of

the tumor environment are not recapitulated in vitro. In the second case, growth of

tumor cells on a two dimensional surface, the presence of high concentrations of growth

factors, high O2 levels and lack of inhibitory cell-cell contacts with surrounding normal

cells could decrease the sensitivity of cells to resveratrol. These optimized growth

conditions for tumor cells in culture may necessitate higher drug concentrations in vitro.

Reports of the relevant serum levels of resveratrol are further complicated by

recent studies and unpublished observations indicating that resveratrol metabolites do

not inhibit tumor cell growth (28, 29) and Subramanian unpublished. This suggests that

glucuronidation and sulfation not only target the drug for removal from the body, but

also mitigate its anti-tumor activity. Minor conversion back to resveratrol by local

sulfatases, for example, would not compensate for this rapid decrease in activity. To

increase the potency of resveratrol treatment, it will be necessary to increase the

amount of unmetabolized compound at the tumor site. A major obstacle to attaining this

goal systemically is the low solubility of the drug. Future research should be directed






towards new formulations and analogs that increase its bioavailability and delay its

metabolism (Fig. 2).

The bioavailability conundrum also brings up the issue of resveratrol’s ability to

differentiate between normal cells and cancerous cells in animal models. At low

concentrations where there is no evidence of cell death either in normal or tumor tissue,

there is nevertheless an anti-tumor effect of the drug leading to tumor inhibition. At

higher local concentrations where there is significant evidence for cell death in tumor

tissue, the surrounding normal tissue remains unaffected (11). This discrimination

between normal and tumor cells could arise due to differences in: 1) the cellular uptake

of resveratrol; 2) the expression of cellular targets or 3) a differential ability to modify

resveratrol into less active forms. Studies of these processes are necessary to

understand the non-toxic nature of resveratrol.

V. Consistencies between disease processes

Can the findings from resveratrol research done in the realm of cancer be

generalized to cardioprotection / life prolongation / diabetes prevention / and vice

versa?

Resveratrol has a wide variety of pharmacological activities making its study

relevant to several diseases such as cancer, cardiovascular and neurodegenerative

diseases and diabetes. However, it is not always clear how the same drug causes

different or even opposite effects in different cell types and in different diseases. For

example, resveratrol causes cell death in tumor cells but is effective as an anti-apoptotic






or protective agent in nerve cells (30). Similarly, resveratrol is anti-angiogenic in certain

tumor models yet pro-angiogenic in the case of myocardial infarction (31, 32).

Resveratrol increases mitochondrial health in muscle cells while it induces

mitochondria-mediated apoptosis in tumor cells (33). How might this occur? While most

direct targets of resveratrol may be expressed in a variety of different cell types albeit at

different levels, intracellular signaling networks will be different depending on the cell

type and cell status (non-dividing, dividing, tumorigenic) leading to different outcomes

following initial target interaction.

Epidemiologic studies suggest a correlation between diabetes and cancer risk.

Resveratrol has been shown to exhibit anti-diabetic effects. A recent report of the use of

the anti-diabetic drug metformin in cancer treatment suggests that resveratrol’s anti-

diabetic effects may be relevant to its anti-tumor effects as well (32, 34). Therefore,

findings of resveratrol action will be of potential cross-disease importance. As different

molecular pathways are elucidated in greater detail and are incorporated into larger

networks, it will become increasingly apparent how addition of a drug like resveratrol

can still be effective in very disparate diseases.

Many of these diseases are prevalent simultaneously, particularly in elderly

patients. Based on current literature, it would appear that resveratrol could ameliorate

secondary conditions even as it is used to treat a primary disease. However, it is

necessary to carry out comprehensive clinical trials on both the preventive potential as

well as curative efficacy of resveratrol in different diseases before such claims can be

validated.






VI. Market Claims

What can we tell the public?

Resveratrol has great potential as an anti-cancer drug either as primary or as

adjuvant therapy. One of its major benefits is a lack of toxicity at doses needed for its

anti-tumor effect. Toxicity remains one of the major concerns with current

chemotherapies, and resveratrol therefore may allow for lowering of the doses of toxic

compounds while maintaining or enhancing the anti-tumor efficacy. Furthermore, there

have been no reports of acquired resistance to resveratrol during treatment.

At this time no clinical trials have been completed to indicate that resveratrol will

be effective as an anti-cancer treatment in humans. All the studies to date have been

performed in tissue culture models or animal models. These studies show a strong anti-

tumor effect of resveratrol with oral dosing and the potential for even higher efficacy if

doses can be further increased by overcoming delivery and solubility obstacles.

However, as has been shown with numerous other potential drugs, results from animal

studies do not necessarily extrapolate to humans. Therefore great caution has to be

taken not to raise expectations of success in using this compound as an anti-cancer

agent before clinical trials have been successfully conducted. Even as these trials

proceed, the concerns raised in previous sections should be adequately addressed in

order to maximize resveratrol’s potential as a non-toxic anti-cancer drug. Currently, one

trial is underway in colon cancer patients with trials for a number of other types of

cancer proposed. Ultimately, based on the results of the clinical trials, the FDA alone

has the authority to approve the use of resveratrol in cancer therapy in the United






States. In addition, its antioxidant effects currently being tested in other clinical trials,

may lead to its use as an approved chemopreventive agent or in combination with other

anti-cancer drugs. Again, the non-toxic aspects of resveratrol also make it a likely

candidate for neoadjuvant or adjuvant treatment.

Nevertheless, resveratrol is already available as a nutritional supplement.

Because nutraceuticals are more loosely regulated by the FDA, much stronger claims

regarding resveratrol’s benefits to human health and disease are touted by this industry.

Since those afflicted with cancer are desperate for a cure and are distrustful of current

chemotherapy, many turn to nutraceutical products drawn by the promise of a

successful ‘natural’ treatment. Unfortunately many of these claims regarding resveratrol,

go well beyond current research results leading to false hopes in patients. These

expectations are increased further by reports in newspapers and on television

promoting resveratrol’s anti-cancer and anti-aging properties along with a host of other

health benefits. All these claims are yet to be scientifically proven in humans. It is not

surprising that companies with a commercial interest in resveratrol may oversell its

qualities in order to promote their product. However, it is important that researchers

provide accurate information through peer-reviewed publications and to the public via

news outlets and other avenues indicating the limitations of the studies performed to

date. As results of human trials become known, we will know whether the initial

promises can be fulfilled.






Acknowledgements

We thank Chue Vang for excellent technical assistance. This work was supported by

funds from the American Institute for Cancer Research (PvG), the Mandelbaum

Therapeutics Initiative (ASP) and the Retina Research Foundation (ASP is the M.D.

Matthews RRF Professor).

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Figure Legends

Fig 1: Resveratrol-Induced Ca2+ -Mediated Apoptosis . Resveratrol activates the

inositol triphosphate receptor (IP3R) either through an extracellular receptor or through

an intracellular target resulting in release of ER calcium reflected in a rapid rise in

intracellular calcium ([Ca2+]i). The subsequent calcium uptake by the mitochondria

causes mitochondrial membrane depolarization (Ψm), increase in reactive oxygen

species production (ROS), release of cytochrome c and smac/diablo activating the

intrinsic apoptotic pathway via caspases-9 and -3. Mitochondrial calciuminduced-

calcium-release (mCICR) results in the activation of calpain that then cleaves various

substrates including calcium exchangers and pumps like PMCA1 leading to additional

elevation of intracellular calcium further stimulating cell death.

Fig 2: Translation of Resveratrol to the Clinic. Successful translation of resveratrol

to an approved chemotherapy drug will necessitate new, more soluble formulations to

improve resveratrol bioavailability and delay metabolism to achieve significant

improvements in anti-tumor efficacy.




© 2010 American Association for Cancer Research

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Extracellularreceptor pumps/exchangers

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© 2010 American Association for Cancer ResearchC R h

Existingformulations

Newformulation

Low solubilityand fast resveratrol

metabolism

High solubility andslower resveratrol

metabolism

Low resveratrolbioavailability

Unmetabolizedresveratrol

High resveratrolbioavailability

Unmetabolizedresveratrol

MetabolitesMetabolites

Tumor inhibition Tumor regression

InactiveInactive

IV, local

Resveratrol

OH

OH

HO

Diet Supplement Pills




Published OnlineFirst November 2, 2010.Clin Cancer Res Lalita Subramanian, Sherry Youssef, Saswati Bhattacharya, et al. discussionResveratrol: challenges in translation to the clinic - A critical

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resveratrol: challenges in translation to the clinic · 29/10/2010 · now that 60 minutes,...

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